UBC Theses and Dissertations
Macrophage interaction with Pseudomonas aeruginosa Kluftinger, Janet Louise
The interactions of macrophages with Pseudomonas aeruginosa were studied. Five monoclonal antibodies specific for porin protein F were tested for their ability to opsonize P. aeruginosa for complement-independent phagocytosis by unelicited mouse peritoneal macrophages, human peripheral blood monocytes and mouse macrophage cell line P388[sub D1]. All five antibodies significantly increased the level of bacterial uptake over that obtained with the non-opsonic controls. The relative effectiveness of the different antibodies was approximately the same in all cell types indicating that the P388[sub D1] cells can be used as a model for normal macrophages. Of the four monoclonal antibodies directed against similar epitopes of protein F, the three IgGl monoclonal antibodies were substantially more opsonic than the one IgG2a isotype. P. aeruginosa cytotoxin and periplasmic contents caused a significant reduction in antibody-mediated phagocytosis of P. aeruginosa. Phagocytosis was restored upon pre-incubation with anti-cytotoxin serum. Both cytotoxin and periplasmic contents caused depolarization of the P388[sub D1] cell membrane, as demonstrated using a polarization-sensitive fluorescent probe. These data indicated that P. aeruginosa cytotoxin was localized in the periplasm and had the potential to inhibit macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane. Monoclonal antibodies directed against protein F were also capable of enhancing phagocytosis of in vivo-grown P. aeruginosa. P. aeruginosa cells taken directly from the in vivo growth system were significantly more susceptible to macrophage phagocytosis than were the same cells after being washed in buffer. The phagocytosis-promoting factor could be isolated from the supernatant of in vivo-grown bacteria and was determined to be fibronectin. Data indicated that promotion by fibronectin of non-opsonic phagocytosis was mediated by direct activation of the macrophages. The tetrapeptide arginine-glycine-aspartate-serine in the eukaryotic cell binding domain of fibronectin was demonstrated to be the macrophage-activating region. Phagocytosis of a mutant P. aeruginosa strain lacking surface pili could not be enhanced by fibronectin. Furthermore, exogenously added Pseudomonas pili was capable of abrogating the enhanced phagocytosis of the wild type strain observed with fibronectin-activated macrophages. It was concluded that Pseudomonas pili were the bacterial ligands required for attachment to fibronectin-activated macrophages in the initial stages of non-opsonic phagocytosis.
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